Our long-term goal is to understand the mechanisms by which molecular motors drive directional traffic along cytoskeletal filaments in the crowded cellular milieu. Specifically, we aim to understand how kinesin motors, whose biophysical characteristics have been well-studied in vitro, drive the transport of organelles and vesicles along microtubule tracks in vivo. To do this requires new techniques for nanoscale tracking of the distribution, activity and interactions of individual molecules in the cytoplasm. By combining techniques from the physical, chemical, biological and mathematical sciences, we have developed new methodologies, based on genetic labeling with fluorescent proteins and total internal reflection fluorescence (TIRF) microscopy, that provide the first analysis of single Kinesin-1 motors in the cytoplasm of live cells. Using this system, single molecule imaging in live-cells (SMILe), we have shown that the motility of single truncated Kinesin-1 motors is not hindered by the crowded cellular environment nor upregulated by unknown cellular factors (Biophysical Journal, in press). Understanding how single motors work in vivo is an essential first step to answer cellular questions such as how multiple motors assemble together and integrate their activities to drive membrane trafficking events along crowded microtubules. In Aim 1, we will continue to develop SMILe methodologies for imaging at the nanoscale and use these methods to test other systems (e.g. other kinesins, neuronal cells) and various conditions (e.g. different temperatures and co-expressed accessory proteins). In Aim 2, we will develop two-color SMILe methodologies to characterize the influence of cellular crowding on kinesin motors. In Aim 3, we will use the two-color methodologies to examine the interactions and activities of multiple motors and how they cooperate to drive motility of individual cargoes in live cells. The results of this application will give new insights into the biophysical parameters that control motor protein-based transport. The methods and approaches advanced in this application will be applicable to a wide variety of biological systems (transcription, translation, synaptic transmission, membrane trafficking, etc.) that are driven by the action of a surprisingly low number of molecules. In addition, these results will aid in the design of engineering and diagnostic devices and the development of treatments for neurodegenerative diseases, cancer and viral infection. Project Narrative: The movement of proteins, organelles, and other cellular components is driven by molecular motors. Defects in motor-driven transport have been shown to be associated with neurodegenerative diseases, cancer, and polycystic kidney disease. Understanding how molecular motors function in cells will provide important new targets for therapies aimed at these diseases.